More than 50 years ago it was observed that serial passage of DEN serotype 1 or 2 in mouse brain rapidly selected for neurovirulent mutants that concomitantly exhibited significant attenuation for humans. This rapid coordinate acquisition of mouse neurovirulence and attenuation for humans suggested that the genetic basis of these two phenotypes might be related. In an attempt to investigate this phenomenon we constructed viable chimeric viruses containing the C-PreM-E or PreM-E genes of a neurovirulent dengue virus mutant or chimeras containing one or more amino acid changes present in the neurovirulent mutant on a background of the parental dengue virus sequence. These chimeras were then tested for neurovirulence in suckling mice. When the E gene was derived from the neurovirulent DEN2 mutant and the PreM gene from the parental virus or, alternatively, the E gene contained amino acid changes Glu351-Asp and Glu406-Lys, the resulting virus exhibited mouse neurovirulence. The conservative amino acid substitution of Glu351-Asp probably had a minor effect and it is likely that the latter mutation was responsible for the acquisition of DEN2 mouse neurovirulence. The DEN4 H241 parent and its neurovirulent mutant exhibit three amino acid differences in the E glycoprotein. Two of these changes in the mutant were identified as important determinants of mouse neurovirulence: (1) The substitution of Ile for Thr434, which ablated one of the two conserved glycosylation sites in parental E, yielded a virus that was almost as neurovirulent as the mouse-adapted mutant, and (2) the substitution of Leu for Phe680 in E also yielded a neurovirulent virus, but it was less neurovirulent than the glycosylation mutant. Similar studies were performed to determine the genetic basis for DEN1 mouse neurovirulence. For reasons that are not clear at present, the chimeras containing the C-PreM-E genes of mouse neurovirulent DEN1 MD-1 strain or its higher mouse passage virus did not exhibit greater neurovirulence than the chimera containing corresponding genes of parental DEN1. Other studies with DEN3 demonstrated that it might be possible to create mouse neuroviruent dengue viruses of all four serotypes for evaluation of attenuation as part of a general strategy for vaccine development.